Identification of salmonella

ABSTRACT

A new culture medium for identifying the presence of Salmonella inenterobacteria samples, especially faeces, contains two chromogenic enzyme substrates, one of which is a substrate for α-D-galactosidase, for which Salmonella is positive. The other substrate is one for which Salmonella is negative such as β-D-galactosidase. The substrates are incorporated into an agar medium. Positive and negative results are found to be readily observable where one of the substrates is an esculetin, preferably a cyclohexenoesculetin compound in the presence of ferric ions, which produces a black color, and the other substrate is an indoxyl compound, for instance, a 5-bromo-4-chloro-3-indolyl compound which produces a green colored enzymic reaction product.

This application claims benefit under rule 371 of PCT GB-98/01645 filedJun. 4, 1998 which claims benefit from EP 97/3846.6 filed Jun. 4, 1997.

The present invention relates to processes for identifying the presenceof Salmonella species in a sample, as well as culture media suitable forsuch identification processes.

Members of the genus Salmonella constitute the most important causes offood poisoning in the UK. At present, the only effective means ofdiagnosis involves cultural isolation of the causative organism fromfaeces. This however is not straightforward as specialised media andreagents are required to isolate relatively small numbers of Salmonellaefrom a massive amount of commensal flora in the guts. Selective mediahave been developed for this purpose which rely on the visualisation ofsimple biochemical features such as production of hydrogen sulphide ornon-fermentation of lactose.

A useful review of five plating media for isolation of Salmonellaspecies and a comparison against Hektoen enteric agar, a standardmedium, is described by Dusch et al in J. Clin. Microbial. (1995) 33(4),802 to 804. All but one of the media are solid (standard agarconcentration) whilst one is a semi solid reduced agar concentrationmedium. For the solid media, the compounds which are produced in thepresence of microbial growth are selected so as to be visible to thenaked eye. In order that the visualised compounds are associated withmicrobial colonies, those compounds must be non-diffusible in theculture medium. These media typically test for two different biochemicalcharacteristics of bacterial colonies and the results are such thatpositive and negative results of each of the two tests can be observedwith positive or negative results of the other test. Some of thebiochemical tests observe the activity of specific enzymes by the use ofchromogenic substrates which are uncoloured or non-fluorescent but whichgenerate enzymic reaction products which are coloured or fluorescent andcan hence be observed in the presence of the substrates. Sometimes theenzymic reaction product may react with a further component of theculture medium to generate the visible product, for instance metal ionsor pH indicators, where the reaction product is an acid or base.

It is known to include in the culture medium substrates for twodifferent enzymes which have different enzymic reaction products, eachof which can be observed in the presence of the other (and of each ofthe substrates themselves).

One enzyme substrate which is commonly used in the identification ofSalmonella is a substrate for β-galactosidase. Salmonella is generallynegative for this enzyme activity, but most other members of theEnterobacteriaceae are positive. One β-galactosidase substrate whoseenzymic reaction product is non-diffusible is5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X Gal). Other indoxyland halogenated indoxyl compounds are useful as substrates and havereaction products which are visible and non-diffusible in agar culturemedia.

X-Gal is used as a substrate in Rambach medium, described inter alia inU.S. Pat. No. 5,194,374. It is used in combination with an alkanediol,which is metabolised by Salmonella to form an acid reaction productwhich is visualised by the incorporation of a pH indicator such asneutral red.

In EP-A-0516817, a culture medium for detecting Salmonella comprises achromogenic β-galactosidase substrate and, in addition, glucuronate anda pH indicator. This mixed medium is alleged to be more selective thanRambach medium since almost all Salmonella species tested, but few otherbacterial species ferment glucuronic acid resulting in a lowering of thepH.

In WO-A-94/0 1952, a 5-bromo-4-chloro-3-indolyl compound which is asubstrate for an esterase enzyme is used to identify Salmonellae, whichare positive for such enzymes. The substrate is an ester of aC₇₋₁₀-fatty acid. It is suggested that the medium may be supplemented toeliminate non-Salmonella bacteria, such as using properties relating tocleavage or metabolism of β-galactosides and β-glucosides (for both ofwhich Salmonella is negative).

Rambach medium and the X-gal glucuronic acid combination were found byDusch et al to have less than optimal sensitivities. A further mediumcomprising xylose, lysine and Tergitol 4 has very good sensitivity andspecificity. The culture medium includes the surfactant Tergitol 4 toinhibit Proteus, and determining hydrogen sulphide formation from sodiumthiosulphate in the medium which is visualised by the incorporation offerric ions.

It is known that Salmonella species produce α-galactosidase, but it islikely that that enzyme would be considered a poor marker for Salmonellasince it is produced by many related genera, such as Escherichia,Citrobacter, Klebsiella, Enterobacter and Shigella.

In Acta Microbiol Hung. (1988) 35(4), 389-395 Ketyi, I. discusses theα-galactosidase activity of various species of entero-bacteria includingSalmonella, Shigella and E-coli. He indicated that enzymic activity is ageneral feature of Enterobacteriaceae. He used melibiose, as anindicator of α-galactosidase positive strains. Melibiose is not achromogenic compound.

In WO-A-9630543 a chromogenic β-galactosidase substrate is used incombination with a mixture of sugars including mannitol, with xylose andmelibiose for identifying Salmonella. The sugars are cleaved to formacids and the growth medium contains a pH indicator. However the acidswhich change the pH are products of a series of enzymic reactions on theproduct of sugar metabolism.

James et, al in App. Env. Microbiol. (1996), 62(10) 3868-170 and in J.App. Microbiol.(1997),82, 532-536, describe a new β-galactosidasesubstrate for use in place of X-Gal. The substrate is a derivative ofcyclohexenoesculetin, of which the aglycone released by hydrolysis byβ-galactosidase forms a black-brown complex with ferric ions in themedium. The new substrate, CHE-Gal, gave good correlation with X-Gal,that is high specificity and high sensitivity for detectingβ-galactosidase activity.

These cyclohexenoesculetin substrates and other esculetin derivativesare described further and claimed in WO-A-9741138 (not published at thepriority date of the present invention).

One aspect of the present invention is based on the need for culturemedia which are very sensitive to Salmonella whilst being highlyspecific, thereby minimising subsequent confirmatory tests. These typesof test often need to be carried out with the inadequately specificmedia of the prior art. A second aspect of the invention is based on theprovision of a medium comprising two chromogenic substrates which givesreadily observable results. A visual determination can be easily made ofthe presence and absence of enzymic reaction products of each substrateregardless of the presence or absence of the enzymic reaction product ofthe other substrate.

According to a first aspect of the present invention there is provided anew process in which the following steps are carried out:

1. a sample suspected of containing Salmonella bacteria is cultured inthe presence of a nutrient,

2. the bacterial culture is contacted with each of two enzymesubstrates,

3. the presence of the enzymic reaction products of each of thesubstrates is accessed after step 2 to determine whether or not growthof Salmonella species has taken place, in which the first substrate is asubstrate for an enzyme for which Salmonella is negative, the processbeing characterised in that the second substrate is a substrate forα-galactosidase and in that both substrates are chromogenic.

The present inventors believe that it is the first time thatα-galactosidase has been used as a marker for Salmonella using anα-galactosidase specific chromogenic substrate, that is a substrate forwhich the enzymic product of the reaction in the presence ofα-galactosidase is chromogenic without being subjected to furtherenzymic reactions. Thus, the inventors have recognised the utility ofcombining β-galactosidase and α-galactosidase as markers for detectingSalmonella species. The method is useful for carrying out the usualtests to identify Salmonella. It is not necessary for there to be anyspecific expectation of Salmonella presence in a clinical sample testedin the present invention. Thus the invention is suitable for screeningto exclude Salmonella (giving a negative result) as well as for positivetests.

The enzyme for cleaving the said first substrate, the activity for whichSalmonella is negative, is selected such that a positive result(cleavage) can exclude a large number of Enterobacteriaceae. Salmonellaeare negative for β-glucosidase and a substrate for β-glucosidase couldtherefore be used. Many other enterobacteria are also negative forβ-glucosidase. Best results are achieved where substrates for thatenzyme are used in combination further with other enzyme substrate. Suchother substrates would be selected to help distinguish Salmonella fromsuch other β-glucosidase negative species. Most conveniently the firstsubstrate is selected to be cleavable by β-galactosidase. The substratetherefore should preferably be a derivative of β-D-galactopyranoside.

Although the invention may be used in a panel of biochemical tests, eachof which is carried out in an individual container, on a singlebacterial colony, it is preferred that the process is used for samplescontaining a mixture of bacterial species which are cultured together ona body of culture medium in a single container. The culture medium ispreferably a solid (gelled) medium, most conveniently based on agar.Other conventional support materials for bacterial culturing can beused.

The sample, as mentioned above, preferably contains a mixture ofbacterial species. It may be a direct sample, inoculated using asuitable technique onto the culture medium. Thus it may be a sample offood, water, or bodily fluid of a patient, usually blood, urine or, mostpreferably faeces. Alternatively a direct sample may, prior to carryingout the process, be enriched by inoculating the direct sample into anenrichment broth and culturing the broth for a period of time, forinstance 24 hours, before inoculating a portion of the bacterial cultureonto the culture medium for the process of the invention. The enrichmentmedium is selected so as to favour the growth of Salmonella species overother common enterobacteria such as E.coli and Proteus. Suitableenrichment media are, for instance, tetrathionate or selenite broths.

The medium in which the first culturing step of the invention is carriedout preferably includes components which favour growth of Salmonella.Thus the medium may contain known inhibitors of other enterobacterialgrowth such as brilliant green, bile salts or desoxycholate sodium salt.

Where the enzyme activity for which Salmonella is negative isβ-galactosidase, the first step of the process of the present inventionis preferably carried out in the presence of a β-galactosidase promoterof known type, for instance lactose or, preferably,isopropyl-β-D-thiogalactopyranoside.

In the process the enzyme substrates are each chromogenic. In thisspecification, the term chromogenic encompasses fluorogenic. The enzymicreaction products or each substrate are preferably directly visible, forinstance as coloured compounds, optionally in the presence of othercomponents such as metal ions, preferably by the naked eye in visiblelight. Alternatively the reaction products may be detectablespectrophotometrically, by observing absorbed radiation of anypredetermined wavelength, or fluorometrically by observing fluorescence.

Alternatively the direct enzyme reaction products may be visible afterfurther chemical, non-enzymic reaction.

It is preferred that the direct product of the enzymic cleavage isdetectable without further chemical reaction, since such furtherreactions may be non-specific and cause false readings. The inventiondoes not include the use of pH indicators to identity the presence ofthe cleavage product.

Where both substrates are in physical admixture in the same body ofculture medium, the enzymic reaction products of the two substrates mustbe different compounds, at least one of which should be detectable inthe presence of the other and in the presence of both the substratesthemselves. The other reaction product might be masked by the first, orbe visible in its presence. Thus any combination of positive andnegative reaction can be observed in the reaction medium.

Although the step of contacting the substrates with the culturedbacteria may take place after culturing has been carried out for aperiod of time, and in a step in which no further bacterial growth ormetabolism takes place, preferably culturing takes place in the presenceof the enzyme substrates. Thus the substrates are incorporated into theculture medium at the beginning of the culturing step 1 of the process.The substrates should, consequently, be non-toxic for bacteria, or atleast for Salmonella, allowing growth of, especially, Salmonella, totake place.

The present inventors have discovered that a particularly usefulcombination of enzymic substrates comprises a substrate which generatesan enzymic reaction product which is an indoxyl compound, including ahalogen substituted compound, and a second substrate which is anesculetin, especially a 3,4-cyclohexenoesculetin compound. Where thelatter substrate is used, during or after contacting of the culturedbacteria with the substrate ferric ions should be contacted with themedium. This leads to generation of a brown black colour with theenzymic reaction product of such a substrate.

According to a second aspect of the invention there is provided aprocess in which a bacterial sample is cultured on a solid medium whichcomprises, in admixture, two chromogenic enzyme substrates, the enzymicreaction products of which are substantially non-diffusible in the solidmedium, are capable of being detected optically in the presence of therespective substrates, and are different compounds, in which one of thesubstrates is an indoxyl compound and characterised in that the other ofthe substrates is an esculetin compound, preferably a3,4-cyclohexenoesculetin compound.

Preferably one of the enzyme substrates is a substrate for aglycosidase, for instance β-galactosidase, α-galactosidase orβ-glucosidase. Preferably one of the substrates is a substrate for adifferent glycosidase enzyme, although may alternatively be an esterasesubstrate. Preferably both substrates are for different glycosidaseenzymes. Most preferably one substrate is for α-galactosidase and theother is a substrate for β-galactosidase.

The esculetin substrate is substituted at the 6- or, preferably,7-hydroxyl by a glycoside. Substituted 3,4-cyclohexenoesculetincompounds which produce non-diffusible complexes with metal ions, forinstance, ferric ions, may also be used. Thus the cyclohexene ring maybe substituted, or the coumarin ring system may be substituted, by oneor more substituents.

The esculetin compound suitably has the general formula

wherein

each of R¹ and R² independently represents a hydrogen or a halogen atomor another group which does not interfere with subsequent ironchelation;

each of R³ and R⁴ independently represents a hydrogen atom or a (C₁-C₈)alkyl or (C₆-C₁₀) aryl (C₁-C₈) alkyl or an optionally modifiedcarboxyl-bearing group of the general formula —CH₂(CH₂)_(n)COX, where nis a number from 0 to 3 and X represents a hydroxyl group or anotherhydrophilic group,

and, R³ may alternatively represent an acyl group of the general formula—COR, in which R represent a (C₁-C₈)alkyl, (C₆ or C₁₀)aryl(C₁-C₈)alkylor (C₅-C₈) cycloalkyl group,

provided that R³ and R⁴ between them contain at least three carbonatoms;

or R³ and R⁴ together with the carbon atoms to which they are attachedform a (C₅-C₈) cycloalkene ring; and

one of Y and Z represents the enzymatically cleavable group and theother of Y and Z represents a hydrogen atom;

or a suitable salt or hydrate thereof.

Hereafter in this specification the term “compound” includes “salt” or“hydrate” unless the context requires otherwise.

As used herein the term “halogen” or its abbreviation “halo” meansfluoro, chloro, bromo and iodo.

The expression “atom or group which does not interfere with ironchelation” refers to the fact that one of the principle means ofdetection of aglycones of general formula I is by chelation by means ofhydroxyl groups at the 6 and 7 positions of the coumarin ring system.Groups which do not interfere with this chelation may be substituted atR¹ and/or R². Examples include hydrogen, hydroxyl, halogen or (C₁-C₆)alkyl. The halogen atom may be a fluorine atom or a chlorine atom andthe lower alkyl group may be methyl, ethyl, propyl, butyl or benzyl.

As used herein the term “(C₁-C₈)alkyl” refers to straight chain orbranched chain hydrocarbon groups having from one to eight carbon atoms.Illustrative of such alkyl groups are methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, heptyland octyl. From one to four carbon atoms may be preferred.

As used herein the term “(C₁-C₁₀)alkyl” refers to straight chain orbranched chain hydrocarbon groups having from one to ten carbon atoms.Illustrative of such alkyl groups are methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl,heptyl, octyl, nonyl and decyl. From one to six carbon atoms may bepreferred.

The term “(C₆ or C₁₀)aryl” includes phenyl and naphthyl.

As used herein, the term “(C₅-C₈) cycloalkene ring” refers to analicyclic ring having from 5 to 8 atoms and having in addition one ormore double bonds. Illustrative of such cycloalkenyl groups arecyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.

In compounds of this invention, the presence of an asymmetric carbonatom gives rise to enantiomers. The presence of several asymmetriccarbon atoms give rise to diastereoisomers, each of which consists oftwo enantiomers, with the appropriate R or S steriochemistry at eachchiral centre. The invention is understood to include all suchdiastereoisomers, optically active enantiomers and mixtures thereof.

The term “suitable salt” refers to a salt prepared by contacting acompound of formula I with an acid or base whose counterpart ion doesnot interfere with the intended use of the compound. Examples includethe sodium salt or magnesium salt of a phosphate derivative or the saltformed from a primary, secondary or tertiary amine where the compound orgeneral formula I is a carboxylic acid. An example of a primary aminesalt can be the cyclohexylammonium salt, a suitable secondary amine saltmay be the piperidine salt and a tertiary amine salt may be thetriethylamine salt.

Preferred compounds of general formula I include those in which,independently or in any compatible combination:

R₁ is chlorine or, preferably hydrogen;

R² is chlorine or, preferably hydrogen;

R³ is (C₁-C₄)alkyl, particularly butyl, or benzyl;

R⁴ is (C₁-C₄)alkyl; or, —CH₂(CH₂)_(n)COX, where n is a number from 0 to3 and X represents a hydroxyl group or one of the following hydrophillicgroups, namely:

—NHCH₂CONHCH₂CO₂H

—NHCH₂CONHCH₂CONHCH₂CO₂H

—NHCHCH₂CONH₂

R³ and R⁴ together with the carbon atoms to which they are attached forma (C₅-C₈)cycloalkene ring, preferably a cyclopentenyl or cyclohexenylring;

where R³ is —CH₂(CH₂)_(n)COX, where n is a number from 0 to 3, then thegroup X is as previously defined,

the enzymatically cleavable group represented by Y or Z is an α- or,preferably, β-linked sugar residue such as β-D-glucose, β-D-galactose,β-D-xylose, β-D-glycuronic acid or N-acetyl-β-D-glucosamine. Sugarresidues derived from galactose, especially β-D-galaclopy-anosides, aremost preferred compounds.

Compounds in which R³ and R⁴ together with the carbon atoms to whichthey are attached form a cyclopentene or a cyclohexene ring areespecially preferred.

A preferred compound of general formula (I) is:

3,4-cyclohexenoesculetin-β-D-galactoside,

The enzymic reaction product of a 3,4-cyclohexenoesculetin substrateproduces a brown black complex in the presence of ferric ion. Theenzymic reaction product of a 5-bromo-4-chloro-3-indolyl compoundproduces a green or blue colour in the presence of oxygen. Other indoxylderivatives are available, which have different substituents so as togenerate a different coloured reaction product, for instance which ismagenta, rose, blue, salmon red, and any of these can be used in placeof the 5-bromo-4-chloro-3-indolyl compound. A bacterial colony which ispositive for the enzyme which cleaves the esculetin substrate generatesa black colour in the presence of ferric ions. Colonies which arepositive for the enzyme which cleaves the 5-bromo-4-chloro-3-indolylsubstrate produce a green colour. Colonies which are positive for bothenzymes can be distinguished from colonies which are positive for theenzyme of which the indolyl compound is a substrate but which arenegative for the other enzyme or negative for both. The reaction productof the esculetin substrate masks the reaction product of the indoxylsubstrate, however, so that colonies which are positive for both enzymescannot necessarily be distinguished from those which are positive onlyfor the enzyme of which the esculetin compound is a substrate.

The esculetin substrate is generally present in a concentration of about200 to 500 mg/l in the agar medium, more preferably about 300 mg/l. Theindoxyl substrate is present in amounts of up to 300 mg/l, although itis generally unnecessary to use concentrations higher than 100 mg/l. Theamount is usually at least 35 mg/l, for instance about 70 mg/l. Thisconcentration of ferric ions is usually about 400 to 1000 mg/l (based onferric ammonium citrate) for instance about 500 mg/l.

Preferably the method of the second aspect of the present invention isfor identifying the presence of Salmonella. The culturing of thebacteria is therefore preferably carried out in the presence of aninhibitor of other enterobacteria and/or a promoter for β-galactosidase.

According to a further aspect of the invention there is provided a newcomposition for use in the culturing of bacteria which comprises inadmixture a first chromogenic enzyme substrate for β-galactosidase and asecond chromogenic enzyme substrate, the substrates being selected suchthat the enzymic reaction products of the two enzymes are differentcompounds and is characterised in that the second substrate is asubstrate for α-galactosidase.

The new composition of this aspect of the invention is suitable for usein the process of the first aspect of the invention. Preferably thecomposition contains other components suitable for carrying out theculturing step of the bacteria, and thus contains one or more nutrientsfor bacterial growth, and preferably a support substance, for instance agelling substance such as agar. Preferably the composition is in a dry,hydratable form whereby it can be hydrated to form a ready-to-useculture medium. The medium preferably contains the other componentsuseful in the culture medium as described above in connection with theprocess.

According to a further aspect of the invention there is provided a newcomposition for use in the culturing of bacteria comprising in admixturea first chromogenic enzyme substrate which is an indoxyl compound and asecond chromogenic enzyme substrate, and is characterised in that thesecond enzyme substrate is an esculetin, preferably a3,4-cyclohexenoesculetin, compound.

In this aspect of the invention, the composition also preferablycontains one or more nutrients for bacterial growth as well as a supportsubstance, for instance a gelling substance such as agar. Thecomposition is preferably in hydratable form and should contain furtherferric salt, which generates the black compound in the presence of theenzymic reaction product of the esculetin compound. Ferric ammoniumcitrate is conveniently used although ferric gluconate or other saltscould be used as alternative sources of ferric ions.

The compositions may be based on selective basal media which inhibitnormal microbial flora and allow selective growth of Salmonella. Knownmedia of this type are bismuth sulphite agar, Brilliant green agar,Hektoen enteric agar and Salmonella/Shigella agar.

Other preferred embodiments of this further aspect of the invention aredescribed above in connection with the novel processes. As mentionedabove, it is believed that this is the first time that α-galactosidasehas been used as a marker for Salmonella in the detection of Salmonellain a mixed sample.

Accordingly in a further aspect of the invention there is provided a newuse of a chromogenic α-D-galactoside enzyme substrate to detect thepresence of Salmonella species in a mixed species sample. Preferably thedetection is carried out by culturing the mixed sample on an agarmedium. Chromogenic α-galactosidase substrates are commerciallyavailable. The enzyme substrate is preferably5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside.

The invention is described further in the following example:

EXAMPLE

The following base culture medium is made up. It encourages the growthof Salmonella at the expense of other enterobacteria by incorporation ofdesoxycholate.

DCA Hynes Base (per liter) Beef extract 5.0 g Balanced peptone No. 1 5.0g Sodium citrate 8.5 g Sodium desoxycholate 5.0 g Agar No. 2 12.0 g Chromogenic Mix (per liter)5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside  70 mg3,4-cyclohexenoesculetin-β-D-galactopyranoside 300 mg Ferric ammoniumcitrate 500 mg Isopropyl-β-D-thiogalactopyranoside  30 mg

All of the above ingredients are dissolved in 1 liter of distilled waterand autoclaved at 116° C. for 10 minutes. The agar is then poured insterile plastic petri dishes and allowed to set.

Evaluation

Members of the Enterobacteriaceae of known identity were obtained inpure culture and inoculated onto the new selective medium. All plateswere incubated at 37° C. for 18 hours and examined for colourproduction. 1020 of these strains were known to be Salmonella and hadbeen consecutively isolated from faeces samples at both the FreemanHospital (120 strains) and the Newcastle Regional Public HealthLaboratory (900 strains). Of the 1020 Salmonella strains, 1016 (99.6%)produced a green colony characteristic of Salmonella. Of the remainingfour strains there were three strains which did not produceα-galactosidase and remained colourless. These were two strains ofSalmonella saint-paul and one strain of Salmonella branderup. Theremaining strain was a β-galactosidase producing Salmonella arizonaewhich consequently produced a black colony.

Of the 300 non-Salmonella, only one strain produced a green colonytypical of Salmonella. This was a highly atypical strain of Escherichiacoli which did not produce β-galactosidase. 39 other strains of E.coliproduced a typical black colony.

From the above results it can be seen that the culture medium includingthe substrates for α-galactoside and β-galactoside is extremelysensitive (99.6%) but still highly specific (99.9%) for the detection ofSalmonella. Furthermore the results are easy to read.

What is claimed is:
 1. A process of analyzing a sample suspected ofcontaining Salmonella bacteria to determine whether such bacteria arepresent in which the following steps are carried out: a. culturing thesample in the presence of a nutrient to form a bacterial culture, b.contacting the bacterial culture with a first and a second enzymesubstrate, c. assessing the bacterial culture after step b for thepresence of the enzymic reaction products of each of said substrates,and d. determining from the assessment of step c whether or not growthof Salmonella species has taken place, wherein said first substrate is asubstrate for an enzyme for which Salmonella is negative, said secondsubstrate is a substrate for α-galactosidase, and both first and secondsubstrates are chromogenic.
 2. A process according to claim 1 in whicheither the first or the second substrate is an indoxyl compound.
 3. Aprocess according to claim 2 in which the indoxyl compound is a5-bromo-4-chloro-3-indolyl compound.
 4. A process according to claim 1in which either the first or the second enzyme substrate is a esculetincompound.
 5. A process according to claim 4 in which the esculetincompound is a 3,4-cyclohexenoesculetin compound.
 6. A process accordingto claim 5 in which the esculetin compound has the general formula I

wherein each of R¹ and R² is independently selected from the groupconsisting of hydrogen, halogen atoms hydroxyl, (C₁₋₆)alkyl and benzylgroups; R³ is selected from the group consisting of acyl groups of thegeneral formula —COR, in which R is selected from the group consistingof (C₁-C₈) alkyl, (C₆ and C₁₀)aryl (C₁-C₈) alkyl and (C₅-C₈) cycloalkylgroups, R⁴ is selected from the group consisting of hydrogen atoms,(C₁-C₈) alkyl, (C₆ and C₁₀) aryl (C₁-C₈) alkyl groups, optionallymodified carboxyl-bearing group of the general formula —CH₂(CH₂)_(n)COX,where n is a number from 0 to 3 and X represents a hydroxyl group oranother hydrophilic group provided that R³ and R⁴ between them containat least three carbon atoms; or R³ and R⁴ together with the carbon atomsto which they are attached form a (C₅-C₈) cycloalkene ring; and one of Yand Z represents an enzymatically cleavable group and the other of Y andZ represents a hydrogen atom; or a suitable salt or hydrate thereof. 7.A process according to claim 6 in which R³ and R⁴, together with thecarbon atoms to which they are attached, form a C₅₋₈-cycloalkene ring.8. A process according to claim 7 in which R³ and R⁴, together with thecarbon atoms to which they are attached, form a cyclohexene ring.
 9. Aprocess according to claim 1 in which one of the enzyme substrates is5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside.
 10. A processaccording to claim 1 in which one of the enzyme substrates is3,4-cyclohexenoesculetin-β-D-galactopyranoside.
 11. A process accordingto claim 1 in which the sample which is cultured containsenterobacteria.
 12. A process according to claim 11 in which the samplewhich is cultured is derived from an enrichment process in which adirect sample is cultured in an enrichment broth.
 13. A processaccording to claim 11 in which the sample which is cultured in theprocess is a direct non-enriched sample.
 14. A process according toclaim 11 in which the direct sample is of faeces.
 15. A processaccording to claim 1 in which the sample is cultured in the presence ofan inhibitor of enterobacteria other than Salmonella.
 16. A processaccording to claim 1 in which the sample is cultured on a solid medium.17. A process according to claim 16 in which the culture medium on whichculturing takes place contains said first and second enzyme substrates,the enzyme substrates being selected such that the enzymic reactionproduct of each of them is a different compound and is substantiallynon-diffusible in the culture medium.
 18. A process according to claim17 in which one of the substrates is an indoxyl compound and the otheris an esculetin compound.
 19. A process according to claim 18 in whichthe indoxyl compound is a 5-bromo-4-chloro-3-indolyl compound.
 20. Aprocess for culturing and detecting bacteria in a sample in which thebacterial sample is cultured on a solid medium which comprises, inadmixture, two enzyme substrates, the enzymic reaction products of eachof which are substantially non-diffusible in the solid medium, which aredetected optically in the presence of the respective substrates, and aredifferent compounds, wherein one of the substrates is an indoxylcompound and the other of the substrates is an esculetin compound.
 21. Aprocess according to claim 20 in which the esculetin compound is a3,4-cyclohexenoesculetin.
 22. A process according to claim 20 in whichthe esculetin compound has the general formula I

wherein each of R¹ and R² is independently selected from the groupconsisting of hydrogen, halogen atoms hydroxyl, (C₁₋₆)alkyl and benzylgroups; R³ is selected from the group consisting of acyl groups of thegeneral formula —COR, in which R is selected from the group consistingof (C₁-C₈) alkyl, (C₆ and C₁₀)aryl (C₁-C₈) alkyl and (C₅-C₈) cycloalkylgroups, R⁴ is selected from the group consisting of hydrogen atoms,(C₁-C₈) alkyl, (C₆ and C₁₀) aryl (C₁-C₈) alkyl groups, optionallymodified carboxyl-bearing group of the general formula —CH₂(CH₂)_(n)COX,where n is a number from 0 to 3 and X represents a hydroxyl group oranother hydrophilic group provided that R³ and R⁴ between them containat least three carbon atoms; or R³ and R⁴ together with the carbon atomsto which they are attached form a (C₅-C₈) cycloalkene ring; and one of Yand Z represents an enzymatically cleavable group and the other of Y andZ represents a hydrogen atom; or a suitable salt or hydrate thereof. 23.A process according to claim 22 in which R³ and R⁴, together with thecarbon atoms to which they are attached form a C₅₋₈ cycloalkene ring.24. A process according to claim 23 in which R³ and R⁴, together withthe carbon atoms to which they are attached, form a cyclohexene ring.25. A process according to claim 20 in which the indoxyl compound is5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside.
 26. A processaccording to claim 25 in which the esculetin compound is3,4-cyclohexenoesculetin-β-D-galactopyranoside.
 27. A process accordingto claim 20 in which the esculetin compound is3,4-cyclohexenoesculetin-β-D-galactopyranoside.
 28. A process fordetecting the presence of Samonella in a mixed sample suspected ofcontaining Salmonella species including the steps of: cutluring themixed sample in the presence of a chromogenic α-galactosidase substrateto produce a culture product, and optically analyzing the cultureproduct to detect the enzymic reaction product of the chromogenicα-galactosidase substrate, whereby a positive result is used as anindicator of the presence of Salmonella growth.
 29. Process according toclaim 28 in which the substrate is an indoxyl compound.
 30. A processaccording to claim 29 in which the substrate is5-bromo-4-chloro-3-indoxyl-α-D-galactopyranoside.
 31. A processaccording to claim 30 in which the esculetin compound has the generalformula I

wherein each of R¹ and R² is independently selected from the groupconsisting of hydrogen, halogen atoms hydroxyl, (C₁₋₆)alkyl and benzylgroups; R³ is selected from the group consisting of acyl groups of thegeneral formula —COR, in which R is selected from the group consistingof (C₁-C₈) alkyl, (C₆ and C₁₀)aryl (C₁-C₈) alkyl and (C₅-C₈) cycloalkylgroups, R⁴ is selected from the group consisting of hydrogen atoms,(C₁-C₈) alkyl, (C₆ and C₁₀) aryl (C₁-C₈) alkyl groups, optionallymodified carboxyl-bearing group of the general formula —CH₂(CH₂)_(n)COX,where n is a number from 0 to 3 and X represents a hydroxyl group oranother hydrophilic group provided that R³ and R⁴ between them containat least three carbon atoms; or R³ and R⁴ together with the carbon atomsto which they are attached form a (C₅-C₈) cycloalkene ring; and one of Yand Z represents an enzymatically cleavable group and the other of Y andZ represents a hydrogen atom; or a suitable salt or hydrate thereof. 32.A composition for use in the culturing of bacteria which comprises inadmixture a first chromogenic enzyme substrate for β-galactosidase and asecond chromogenic enzyme substrate, the substrates being such that theenzymic reaction products of the two enzymes are different compounds andwherein the second substrate is a substrate for α-galactosidase.
 33. Acomposition according to claim 32 in which one of the substrates is anindoxyl compound.
 34. A composition according to claim 33 in which theindoxyl compound is a 5-bromo-4-chloro-3-indolyl compound.
 35. Acomposition according to claim 33 in which the other of the substratesis an esculetin compound.
 36. A composition according to claim 35 inwhich the esculetin compound is a 3,4-cyclohexenoesculetin compound. 37.A composition according to claim 32 in which one of the substrates is anesculetin compound.
 38. A composition according to claim 37 in which theesculetin compound has the general formula I

wherein each of R¹ and R² is independently selected from the groupconsisting of hydrogen, halogen atoms hydroxyl, (C₁₋₆)alkyl and benzylgroups; R³ is selected from the group consisting of acyl groups of thegeneral formula —COR, in which R is selected from the group consistingof (C₁-C₈) alkyl, (C₆ and C₁₀)aryl (C₁-C₈) alkyl and (C₅-C₈) cycloalkylgroups, R⁴ is selected from the group consisting of hydrogen atoms,(C₁-C₈) alkyl, (C₆ and C₁₀) aryl (C₁-C₈) alkyl groups, optionallymodified carboxyl-bearing group of the general formula —CH₂(CH₂)_(n)COX,where n is a number from 0 to 3 and X represents a hydroxyl group oranother hydrophilic group provided that R³ and R⁴ between them containat least three carbon atoms; or R³ and R⁴ together with the carbon atomsto which they are attached form a (C₅-C₈) cycloalkene ring; and one of Yand Z represents an enzymatically cleavable group and the other of Y andZ represents a hydrogen atom; or a suitable salt or hydrate thereof. 39.A composition according to claim 38 in which R³ and R⁴, together withthe carbon atoms to which they are attached form a C₅₋₈ cycloalkenering.
 40. A composition according to claim 37 in which the esculetincompound is a 3,4-cyclohexenoesculetin compound.
 41. A compositionaccording to claim 32 which contains one or more nutrients for bacterialgrowth and a support substance.
 42. A composition according to claim 41in a dry hydratable form.
 43. A composition according to claim 32 whichcontains an inhibitor of enterobacterial growth selected from the groupconsisting of brilliant green agar, agar containing bile salts, bismuthsulphite agar, agar containing desoxycholate salts, Hektoen enteric agarand Salmonella/Shigella agar.
 44. A composition according to claim 32which contains a promoter of β-galactosidase.
 45. A compositionaccording to claim 44 in which the promoter of β-galactosidase isselected from lactose, isopropyl-β-D-thiogalactopyranoside and mixturesthereof.
 46. A composition for use in the culturing of bacteriacomprising in admixture a first enzyme substrate which is an indoxylcompound and a second enzyme substrate, and wherein the second enzymesubstrate is an esculetin compound.
 47. A composition according to claim46 in which the esculetin compound has the general formula I

wherein each of R¹ and R² is independently selected from the groupconsisting of hydrogen, halogen atoms hydroxyl, (C₁₋₆)alkyl and benzylgroups; R³ is selected from the group consisting of acyl groups of thegeneral formula —COR, in which R is selected from the group consistingof (C₁-C₈) alkyl, (C₆ and C₁₀)aryl (C₁-C₈) alkyl and (C₅-C₈) cycloalkylgroups, R⁴ is selected from the group consisting of hydrogen atoms,(C₁-C₈) alkyl, (C₆ and C₁₀) aryl (C₁-C₈) alkyl groups, optionallymodified carboxyl-bearing group of the general formula —CH₂(CH₂)_(n)COX,where n is a number from 0 to 3 and X represents a hydroxyl group oranother hydrophilic group provided that R³ and R⁴ between them containat least three carbon atoms; or R³ and R⁴ together with the carbon atomsto which they are attached form a (C₅-C₈) cycloalkene ring; and one of Yand Z represents an enzymatically cleavable group and the other of Y andZ represents a hydrogen atom; or a suitable salt or hydrate thereof. 48.A composition according to claim 47 in which R³ and R⁴, together withthe carbon atoms to which they are attached, form a cyclohexene ring.49. A composition according to claim 46 which contains one or morenutrients for bacterial growth and a gelling substance.
 50. Acomposition according to claim 49 in which the gelling substance isagar.
 51. A composition according to claim 50 which contains a promoterof β-galactosidase.
 52. A composition according to claim 51 in which thepromoter of β-galactosidase is selected from the group consisting oflactose, isopropyl-β-D-thiogalactopyranoside and mixtures thereof.
 53. Acomposition for use in the culturing of bacteria comprising agar,5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside and3,4-cyclohexenoesculetin-β-D-galactopyranoside.
 54. A compositionaccording to claim 53 comprising an inhibitor of enterobacterial growthselected from the group consisting of brilliant green agar, agarcontaining bile salts, bismuth sulphite agar, agar containingdesoxycholate salts, Hektoen agar and Salmonella/Shigella agar.
 55. Acomposition according to claim 54 containing a promoter ofβ-galactosidase.
 56. A composition according to claim 55 in which saidpromoter is selected from the group consisting of lactose,isopropyl-β-D-thiogalactopyranoside and mixtures thereof.
 57. Acomposition according to claim 53 containing a promoter ofβ-galactosidase.